Curdlan Production Research Articles

Efficient preparation of β-1,3-glucooligosaccharides by microwave-assisted hydrothermal degradation of curdlan and preliminary exploration of its antibacterial activity

The study focuses on the efficient production of curdlan β-1,3-glucooligosaccharides (CDOS) through microwave-assisted hydrothermal hydrolysis of curdlan. The optimal condition was identified as 170 °C and 30 min, resulting in a curdlan liquefaction yield of over 96%, mainly contains CDOS along with minor amounts of glucose and 5-hydroxymethylfurfural (5-HMF). Oligosaccharides with a low degree of polymerization (DP), specifically DP2 to DP6, were isolated. The yields for DP2 to DP6 were 15.87%, 8.33%, 6.00%, 3.13%, and 1.83%, respectively, with purities over 70%. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) analysis demonstrated that the dense triple helix structure of the crystalline structure was disrupted during degradation. Additionally, CDOS at a concentration of 2.5 mg/mL partially inhibited the growth of Escherichia coli. This study concludes that the combination of microwave-assisted hydrothermal hydrolysis with gel filtration chromatography provides an efficient method for producing and purifying CDOS.

Optimizing Curdlan Synthesis: Engineering Agrobacterium tumefaciens ATCC31749 for Enhanced Production Using Dextrin as a Carbon Source

A key goal in current research on industrial curdlan production is the expansion of carbon sources for fermentation. In this study, a recombinant bacterial strain, sp-AmyAXCC, capable of fermenting and synthesizing curdlan using dextrin as a carbon source, was produced via heterologous expression of IPTG-inducible α-amylase from Xanthomonas campestris NRRL B-1459 in Agrobacterium tumefaciens ATCC31749. External expression of the enzyme was confirmed by western blotting, and the expression levels of exogenous proteins during the fermentation process were monitored. Additionally, the properties of the curdlan product were characterized using attenuated total reflectance-Fourier transform infrared spectroscopy and X-ray diffraction. The recombinant strain produced curdlan at a titer of 30.40 ± 0.14 g/L, gel strength of 703.5 ± 34.2 g/cm2, and a molecular weight of 3.58 × 106 Da, which is 33% greater than the molecular weight of native curdlan (2.69 × 106 Da). In the batch fermentation of sp-AmyAXCC with 12% dextrin as a carbon source, the titer of curdlan was 66.7 g/L with a yield of 0.56 g/g, and a productivity rate of 0.62 g/L/h at 108 h. The results of this study expand the substrate spectrum for Agrobacterium fermentation in curdlan production and provides guidance for further industrialization of curdlan production.

Submerged Fermentation and Kinetics of Newly Isolated Priestia megaterium for the Production of Biopolymer Curdlan

In this study, a curdlan-producing bacterium was isolated from Cow pea soil and identified as Priestia megaterium based on 16 S rRNA sequencing. To identify the most suitable carbon and nitrogen sources for curdlan production, submerged fermentation studies with different sources was carried out. To enhance the curdlan yield, optimization by one-factor-at-a-time approach was conducted. The optimal fermentation media consisted of 15% (w/v) sucrose, 0.1% (w/v) urea, 0.1% (w/v) KH2PO4, 0.04% (w/v) MgSO4·7H2O, trace elements, initial pH of 7.0 with 10% (v/v) inoculum size and agitation speed of 180 rpm. Kinetics of growth, curdlan yield, sucrose and ammonia depletion were studied for a period of 168 h. Maximum curdlan yield (0.31 g/L) was achieved at 96 h of fermentation. At this point, the fermentation media had an optical density of 9.68, biomass concentration of 4.26 mg/mL, and viable count of 2.4 × 104 CFU/mL. Additionally, the maximum percentage consumption of sucrose and ammonia over 168 h of fermentation were 75 and 62.5%, respectively. Finally, the identity of biopolymer curdlan was validated through characterization techniques such as Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM) and Thermogravimetric analysis (TGA). Some characteristic features of curdlan such as the β-1,3-linkage was depicted by the absorption band at 890 cm−1 in FTIR, flaky granules with irregularities as seen in SEM, and thermal degradation between 235 and 350 °C by TGA. To the best of our knowledge, this is the first report on curdlan production from Priestia megaterium.

Genetic Engineering of Agrobacterium Increases Curdlan Production through Increased Expression of the crdASC Genes.

Curdlan is a water-insoluble polymer that has structure and gelling properties that are useful in a wide variety of applications such as in medicine, cosmetics, packaging and the food and building industries. The capacity to produce curdlan has been detected in certain soil-dwelling bacteria of various phyla, although the role of curdlan in their survival remains unclear. One of the major limitations of the extensive use of curdlan in industry is the high cost of production during fermentation, partly because production involves specific nutritional requirements such as nitrogen limitation. Engineering of the industrially relevant curdlan-producing strain Agrobacterium sp. ATTC31749 is a promising approach that could decrease the cost of production. Here, during investigations on curdlan production, it was found that curdlan was deposited as a capsule. Curiously, only a part of the bacterial population produced a curdlan capsule. This heterogeneous distribution appeared to be due to the activity of Pcrd, the native promoter responsible for the expression of the crdASC biosynthetic gene cluster. To improve curdlan production, Pcrd was replaced by a promoter (PphaP) from another Alphaproteobacterium, Rhodobacter sphaeroides. Compared to Pcrd, PphaP was stronger and only mildly affected by nitrogen levels. Consequently, PphaP dramatically boosted crdASC gene expression and curdlan production. Importantly, the genetic modification overrode the strict nitrogen depletion regulation that presents a hindrance for maximal curdlan production and from nitrogen rich, complex media, demonstrating excellent commercial potential for achieving high yields using cheap substrates under relaxed fermentation conditions.

Impact of Using Date Juice as a Carbon Source on Curdlan Produced by a Local Isolate of Agrobacterium leguminum

Agrobacterium species are responsible for the production of the polysaccharide known as curdlan. The curdlan was produced from 10 isolates that were collected from a variety of local sources, including as agricultural soils, root nodules, and plant roots. The isolates were identified by examinations using morphological, microscopic, and biochemical tests. After testing the isolates to see which ones could produce the most curdlan, the A2 isolate finally emerged with a production capacity of 29.2 g.L-1. According to the findings, the production of curdlan was increased using the modified medium that contained date juice at a concentration of 26.4 mL.100 mL-1 of the production medium. The resultant yield was 30.7 g.L-1, which was the highest possible yield. The identification of curdlan was validated by the utilization of FTIR, NMR, and HPLC techniques. The modified medium had the capability of being utilized in the production of curdlan from agricultural waste products.

Production of Curdlan by Agrobacterium sp. DH-2 Using Sugarcane Molasses-Based Medium

Production of Curdlan by Agrobacterium sp. DH-2 Using Sugarcane Molasses-Based Medium

Biofilm-Mediated Fragmentation and Degradation of Microcrystalline Cellulose by Cellulomonas flavigena KU (ATCC 53703).

Cellulomonas flavigena KU (ATCC 53703) produces an extracellular matrix involved in the degradation of microcrystalline cellulose. This extracellular material is primarily composed of the gel-forming, β-1,3-glucan known as curdlan and associated, cellulose-degrading enzymes. In this study, the effects of various forms of nutrient limitation on cellulose attachment, cellular aggregation, curdlan production, and biofilm formation were investigated throughout a 7-day incubation period by using phase-contrast microscopy. Compared to cultures grown in non-limiting media, nitrogen-limitation promoted early attachment of C. flavigena KU cells to the cellulose surface, and cellulose attachment was congruent with cellular aggregation and curdlan production. Over the course of the experiment, microcolonies of attached cells grew into curdlan-producing biofilms on the cellulose. By contrast, bacterial cells grown on cellulose in non-limiting media remained unattached and unaggregated throughout most of the incubation period. By 7days of incubation, bacterial aggregation was ninefold greater in N-limited cultures compared to nutritionally complete cultures. In a similar way, phosphorus- and vitamin-limitation (i.e., yeast extract-limitation) also resulted in early cellulose attachment and biofilm formation. Furthermore, nutrient limitation promoted more rapid and efficient fragmentation and degradation of cellulose, with cellulose fragments in low-N media averaging half the size of those in high-N media after 7days. Two modes of cellulose degradation are proposed for C. flavigena KU, a "planktonic mode" and a "biofilm mode". Similar observations have been reported for other curdlan-producing cellulomonads, and these differing cellulose degradation strategies may ultimately prove to reflect sequential stages of a multifaceted biofilm cycle important in the bioconversion of this abundant and renewable natural resource.

Promoting substrates uptake and curdlan synthesis of Agrobacterium sp. by attenuating the exopolysaccharide encapsulation

During curdlan production by Agrobacterium sp., the secreted exopolysaccharide (EPS) gradually encapsulated Agrobacterium sp., accompanied by cell aggregation, resulted in inhibited substrate uptake and curdlan synthesis. To relieve the EPS encapsulation effect, the shake-flask culture medium was quantitatively supplemented with 2 % to 10 % endo-β-1,3-glucanase (BGN), while obtaining curdlan with a decreased weight-average molecular weight ranging from 18.99 × 104 Da to 3.20 × 104 Da. In a 7-L bioreactor, the 4 % BGN supplement substantially attenuated the EPS encapsulation, resulting in increased glucose consumption and curdlan yield to 66.41 g/L and 34.53 g/L after fermentation of 108 h, which improved 43 % and 67 %, respectively compared with the control. The disruption of EPS encapsulation with BGN treatment accelerated the regeneration of ATP and UTP, resulting in sufficient uridine diphosphate glucose for curdlan synthesis. The upregulation of related genes at the transcription level reveals that the respiratory metabolic intensity, the energy regeneration efficiency, and the curdlan synthetase activity were enhanced. This study presents a simple and novel strategy of relieving the effects of EPS encapsulation on the metabolism of Agrobacterium sp. for the high-yield and value-added production of curdlan, which could be potentially applied in producing other EPSs.

Curdlan production from cassava starch hydrolysates by Agrobacterium sp. DH-2.

Curdlan is an edible microbial polysaccharide and can be used in food, biomedical and biomaterial fields. To reduce the cost of curdlan production, this study investigated the suitability of cassava starch hydrolysates as carbon source for curdlan production. Cassava starch was hydrolyzed into maltose syrup using β-amylase and pullulanase at various enzyme dosages, temperature, time and addition order of two enzymes. The maltose yield of 53.17% was achieved at starch loading 30% by simultaneous addition β-amylase 210 U/g starch and pullulanase 3 U/g starch at 60°C for 9h. Cassava starch hydrolysates were used as carbon source for curdlan production by Agrobacterium sp. DH-2. The curdlan production reached 28.4g/L with the yield of 0.79g/g consumed sugar and molecular weight of 1.26 × 106Da at 96h with cassava starch hydrolysate at 90g/L initial sugar concentration. Curdlan produced from cassava starch hydrolysates was characterized using FT-IR spectra and thermo gravimetric analysis. This work indicated that cassava starch was a potential renewable feedstock for curdlan production.

Optimization and Modeling of Curdlan Production under Multi-physiological-parameters Process Control by Agrobacterium radiobacter Mutant A-15 at High Initial Glucose

Optimization and Modeling of Curdlan Production under Multi-physiological-parameters Process Control by Agrobacterium radiobacter Mutant A-15 at High Initial Glucose

Methionine biosynthesis pathway genes affect curdlan biosynthesis of Agrobacterium sp. CGMCC 11546 via energy regeneration

Curdlan is a water-insoluble exopolysaccharide produced by Agrobacterium species under nitrogen starvation. The curdlan production in the ΔmdeA, ΔmetA, ΔmetH, and ΔmetZ mutants of methionine biosynthesis pathway of Agrobacterium sp. CGMCC 11546 were significantly impaired. Fermentation profiles of four mutants showed that the consumption of ammonia and sucrose was impaired. Transcriptome analysis of the ΔmetH and ΔmetZ mutants showed that numerous differentially expressed genes involved in the electron transfer chain (ETC) were significantly down-regulated, suggesting that methionine biosynthesis pathway affected the production of energy ATP during the curdlan biosynthesis. Furthermore, metabolomics analysis of the ΔmetH and ΔmetZ mutants showed that ADP and FAD were significantly accumulated, while acetyl-CoA was diminished, suggesting that the impaired curdlan production in the ΔmetH and ΔmetZ mutants might be caused by the insufficient supply of energy ATP. Finally, the addition of both dibasic sodium succinate as a substrate of FAD recycling and methionine significantly restored the curdlan production of four mutants. In conclusion, methionine biosynthesis pathway plays an important role in curdlan biosynthesis in Agrobacterium sp. CGMCC 11546, which affected the sufficient supply of energy ATP from the ETC during the curdlan biosynthesis.

Glutamine synthetase gene glnA plays a vital role in curdlan biosynthesis of Agrobacterium sp. CGMCC 11546

Curdlan is a neutral linear exopolysaccharide produced by Agrobacterium spp. under nitrogen-limiting conditions. In this study, we explored the role of glnA in curdlan biosynthesis in Agrobacterium sp. CGMCC 11546. The curdlan production of the ΔglnA strain was impaired, decreasing by 93% compared with that of the wild-type strain after 96 h fermentation. Analysis of fermentation profiles revealed that cell growth and utilization of carbon and nitrogen sources were impaired in the ΔglnA strain. Transcriptome analysis indicated that various of genes involved in curdlan biosynthesis were downregulated after 24 h fermentation in the ΔglnA strain, particularly genes involved in heme synthesis and the electron transport chain, which are essential for energy generation. Metabolomics analysis revealed flavin adenine dinucleotide (FAD) and adenosine diphosphate (ADP) accumulation in the ΔglnA strain, suggesting insufficient energy supply. Furthermore, glnA overexpression led to an 18% increase in the curdlan yield of the ΔglnA mutant compared with that of the wild-type strain after 96 h fermentation. Taken together, the findings demonstrate that glnA plays a vital role in curdlan biosynthesis by supplying ATP via regulating the expression of genes involved in heme synthesis and the electron transport chain.

Production of the Polysaccharide Curdlan by Agrobacterium species on Processing Coproducts and Plant Lignocellulosic Hydrolysates

This review examines the production of the biopolymer curdlan, synthesized by Agrobacterium species (sp.), on processing coproducts and plant lignocellulosic hydrolysates. Curdlan is a β-(1→3)-D-glucan that has various food, non-food and biomedical applications. A number of carbon sources support bacterial curdlan production upon depletion of nitrogen in the culture medium. The influence of culture medium pH is critical to the synthesis of curdlan. The biosynthesis of the β-(1→3)-D-glucan is likely controlled by a regulatory protein that controls the genes involved in the bacterial production of curdlan. Curdlan overproducer mutant strains have been isolated from Agrobacterium sp. ATCC 31749 and ATCC 31750 by chemical mutagenesis and different selection procedures. Several processing coproducts of crops have been utilized to support the production of curdlan. Of the processing coproducts investigated, cassava starch waste hydrolysate as a carbon source or wheat bran as a nitrogen source supported the highest curdlan production by ATCC 31749 grown at 30 °C. To a lesser extent, plant biomass hydrolysates have been explored as possible substrates for curdlan production by ATCC 31749. Prairie cordgrass hydrolysates have been shown to support curdlan production by ATCC 31749 although a curdlan overproducer mutant strain, derived from ATCC 31749, was shown to support nearly double the level of ATCC 31749 curdlan production under the same growth conditions.

Sustainable biosynthesis of curdlan from orange waste by using Alcaligenes faecalis: A systematically modeled approach

This study presents an engineered approach for sustainable biosynthesis of curdlan by Alcaligenes faecalis using orange peels. To confirm the substrate suitability a four step study was organized. Firstly, drying of substrate was carried within temperature range of 60–120 °C, along with the application of moisture diffusion control model. Secondly, fermentation medium was obtained via saccharification and detoxification, releasing highest sugar at 72.34 g/L with phenolics removal of 95–98%. Thirdly, curdlan fermentation was conducted in detoxified orange peel hydrolysate followed by optimization of batch culture fermentation via kinetic modeling using Logistic and Luedeking-Piret equations. In 5 L bioreactor, highest specific growth rate (μm = 0.233/h), highest curdlan production (Pm = 23.24 g/L) and growth associated rate constant (α = 3.403) were achieved. Moreover, the total sugar consumption and conversion rates were 83.27% and 53.20%. Lastly, characterization techniques such as FTIR, NMR, XRD, TGA, HPGPC and EDS were applied to biosynthesized curdlan for qualitative validation.

Optimization and production of curdlan gum using Bacillus cereus PR3 isolated from rhizosphere of leguminous plant

ABSTRACTCurdlan gum is a neutral water-insoluble bacterial exopolysaccharide composed primarily of linear β-(1,3) glycosidic linkages. Recently, there has been increasing interest in the applications of curdlan and its derivatives. Curdlan is found to inhibit tumors and its sulfated derivative possess anti-HIV activity. Curdlan is biodegradable, non-toxic towards human, environment and edible which makes it suitable as drug-delivery vehicles for sustained drug release. The increasing demand for the growing applications of curdlan requires an efficient high yield fermentation production process so as to satisfy the industrial needs. In this perspective, the present work is aimed to screen and isolate an efficient curdlan gum producing bacteria from rhizosphere of ground nut plant using aniline-blue agar. High yielding isolate was selected based on curdlan yield and identified as Bacillus cereus using gas-chromatography fatty acid methyl ester analysis. B. cereus PR3 curdlan gum was characterized using FT-IR spectroscopy, SEM, XRD and TGA. Fermentation time for curdlan production using B. cereus PR3 was optimized. Media constituents like carbon, nitrogen and mineral sources were screened using Plackett–Burman design. Subsequent statistical analysis revealed that Starch, NH4NO3, K2HPO4, Na2SO4, KH2SO4 and CaCl2 were significant media constituents and these concentrations were optimized for enhancement of curdlan production up to 20.88 g/l.

Kinetic analysis of curdlan production by Alcaligenes faecalis with maltose, sucrose, glucose and fructose as carbon sources

Curdlan has wide-ranging benefits in food and pharmaceutical industries for its unique rheological and thermal gelling properties. To analyze the cell growth and curdlan biosynthesis kinetics of Alcaligenes faecalis, the kinetic properties of the curdlan fermentation under different carbon sources conditions (maltose, sucrose, glucose and fructose) were investigated using Logistic and Luedeking-Piret equations. The results demonstrated that curdlan fermentation is partial growth-associated process. With maltose as the sole carbon source, the highest curdlan production (Pm = 39.3 g/L), the maximum specific growth rate (μm = 0.44/h) and the growth-associated rate constant (α = 2.05 g curdlan/g cell) were achieved. In contrast, the fructose was the less desired carbon source in both the cell growth and curdlan production. Further, the results demonstrated that slow-releasing glucose from maltose boosted cell growth and curdlan production.

Function and Regulation of Agrobacterium tumefaciens Cell Surface Structures that Promote Attachment.

Agrobacterium tumefaciens attaches stably to plant host tissues and abiotic surfaces. During pathogenesis, physical attachment to the site of infection is a prerequisite to infection and horizontal gene transfer to the plant. Virulent and avirulent strains may also attach to plant tissue in more benign plant associations, and as with other soil microbes, to soil surfaces in the terrestrial environment. Although most A. tumefaciens virulence functions are encoded on the tumor-inducing plasmid, genes that direct general surface attachment are chromosomally encoded, and thus this process is not obligatorily tied to virulence, but is a more fundamental capacity. Several different cellular structures are known or suspected to contribute to the attachment process. The flagella influence surface attachment primarily via their propulsive activity, but control of their rotation during the transition to the attached state may be quite complex. A. tumefaciens produces several pili, including the Tad-type Ctp pili, and several plasmid-borne conjugal pili encoded by the Ti and At plasmids, as well as the so-called T-pilus, involved in interkingdom horizontal gene transfer. The Ctp pili promote reversible interactions with surfaces, whereas the conjugal and T-pili drive horizontal gene transfer (HGT) interactions with other cells and tissues. The T-pilus is likely to contribute to physical association with plant tissues during DNA transfer to plants. A. tumefaciens can synthesize a variety of polysaccharides including cellulose, curdlan (β-1,3 glucan), β-1,2 glucan (cyclic and linear), succinoglycan, and a localized polysaccharide(s) that is confined to a single cellular pole and is called the unipolar polysaccharide (UPP). Lipopolysaccharides are also in the outer leaflet of the outer membrane. Cellulose and curdlan production can influence attachment under certain conditions. The UPP is required for stable attachment under a range of conditions and on abiotic and biotic surfaces. Other factors that have been reported to play a role in attachment include the elusive protein called rhicadhesin. The process of surface attachment is under extensive regulatory control and can be modulated by environmental conditions, as well as by direct responses to surface contact. Complex transcriptional and post-transcriptional control circuitry underlies much of the production and deployment of these attachment functions.

Influence of Tween-80 on the production and structure of water-insoluble curdlan from Agrobacterium sp.

In order to explore the mechanism by which Tween-80 enhances the production of curdlan produced by Agrobacterium sp., the effects of Tween-80 on the production and structure of curdlan and Agrobacterium sp. were evaluated. Maximum curdlan production (51.94g/L) was achieved when 16g/L Tween-80 was added at the beginning of the cell growth stage. The addition of Tween-80 at higher concentration inhibited cell growth. However, the addition of 16g/L Tween-80 enhanced the production of curdlan with a looser ultrastructure, significantly weakened the envelopment of curdlan on Agrobacterium sp., altered the fine structure of cell membrane, and increased the cell membrane permeability. Moreover, the efficiency of oxygen and mass transport, respiration intensity, UTP regeneration, ATP regeneration, activity of curdlan synthetase, capacity of stress response and energy supply of Agrobacterium sp. were all greatly improved by the addition of Tween-80. These findings demonstrate the mechanisms by which Tween-80 enhances curdlan production and provide a cheap and feasible approach to weaken the envelopment of water-insoluble polysaccharides on bacteria.

Improved curdlan production with discarded bottom parts of Asparagus spear

BackgroundThis work evaluated the improvement of curdlan production of Agrobacterium sp. ATCC 31749 by using culture medium containing juice of discarded bottom part of green Asparagus spear (MJDA). Curdlan production was carried out using Agrobacterium sp. ATCC 31749 in flasks with different volumes of MJDA and its non-juice-adding control (CK) incubated in shaker at 30 °C, 200 rpm rotation for 168 h.ResultsAll MJDA media increased Agrobacterium sp. ATCC 31749 cell mass and enhanced the cells’ ability to utilise sucrose, the carbon source for curdlan biosynthesis, and thereby produced higher concentration of curdlan than CK which is used for commercial production of curdlan. After 168 h of fermentation, 10% MJDA produced 40.2 g/l of curdlan whiles CK produced 21.1 g/l. Curdlan production was increased by 90.4% higher in 10% MJDA than CK. Curdlan produced by 10% MJDA contains 1.2 and 1.5 µg/ml of Asparagus flavonoids and saponins respectively as additives which have wide range of health benefits. The mass of sucrose needed to produce 1.0 g curdlan by Agrobacterium sp. ATCC 31749 in CK is 1.7-fold more than in 10% MJDA.ConclusionThe results strongly revealed that 5–10% MJDA is a good curdlan fermentation media which increase curdlan production yield with cheaper cost of production and simultaneously reduce environmental waste resulting from the large scaled discarded bottom parts of green Asparagus spear during Asparagus production.

Production of Curdlan Grown on Cassava Starch Waste Hydrolysates

Cassava starch waste hydrolysates (CSWHs) with different degrees of polymerisation, i.e., CSWHs-1, CSWHs-2 and CSWHs-3, were prepared through the hydrolysis of cassava starch waste with thermostable a-amylase from Thermococcus sp. HJ21. The prepared CSWHs were then used as a carbon source for curdlan production with Alcaligenes faecalis ATCC 31749. The amount of curdlan produced and the glucosyltransferase activity during curdlan synthesis increased more obviously when CSWHs-2 was used as the carbon source than when glucose was used. Using both carbon sources, the maximum curdlan production was observed at day 5, and the maximum glucosyltransferase activity was observed at day 4. Glucosyltransferase activity decreased thereafter, and biomass continued to increase until the end of the experiment (day 6). Results indicated that the enhanced curdlan production with CSWHs as the carbon source was highly correlated with glucosyltransferase activity.